Simultaneous Bioaugmentation and Biostimulation to Remediate Soil Contaminated by Ship Dismantling in Bangkalan District, Indonesia

Background. High concentrations of total petroleum hydrocarbons (TPH), iron (Fe), and manganese (Mn) were identified in soil samples from two shipyards where vessel dismantling activities take place in Tanjungjati Village, Indonesia, and subjected to bioremediation. Objectives. The aim of the present study was to determine whether the combination of surfactant solution, bioaugmentation (a consortium of Bacillus subtilis and Acinetobacter lwoffii), and biostimulation (nutrient amendment and aeration intermittent) would reduce TPH, Fe, and Mn levels from soil contaminated from ship dismantling activities. Methods. Iron and Mn bioavailability were examined according to the Indonesian technical guidelines for soil chemical analysis with the help of atomic absorption spectrophotometry. The n-hexane solvent soil was extracted using the ultrasonic water bath method for TPH analysis. Results. The highest removal results achieved were TPH (69.62%), Fe (87.10%), and Mn (29%) for Soil 1 samples and elimination of TPH (28.80%), Fe (65.10%), and Mn (57.38%) for Soil 2 samples using a combination of surfactant solution, bioaugmentation, and biostimulation (nutrient amendment and without aeration intermittent). Iron and Mn removal in the controls was higher than in the treated soils, which showed that Fe and Mn could decrease naturally in both contaminated soils. Conclusions. The present study showed that bioremediation using a combination of surfactant solution, a consortium of Bacillus subtilis, and Acinetobacter lwoffii, as well as a nutrient amendment, has the potential to degrade hydrocarbons in contaminated soil. Furthermore, Bacillus subtilis and Acinetobacter lwoffii consortium used for bioaugmentation have the potential to enhance the degradation of hydrocarbons in soil. Competing Interests. The authors declare no competing financial interests.


Introduction
Tanjungjati Village in the Bangkalan district, Madura Island, Indonesia is known for its ongoing ship dismantling activities. Ship dismantling yards take old nonoperational vessels and process them into products that may be sold back into the market chain. The main objective of ship dismantling activities is to obtain steel as a raw material for new ships. The dismantling activities in Tanjungjati Village consist of three stages: ship arrival and administration, pre-cutting (ship cleaning), and cutting. At the pre-cutting stage, the vessel is cleaned of flammable materials such as fuel, paper, rope, and plastic. Subsequently, decoating of the vessel is done by sandblasting to reduce the risk of fire in subsequent stages. The decoating process removes sand and metal particles from the air. Ship body cutting is conducted using the oxy-acetylene technique, which is considered to have lower risks than the use of oxy-liquified petroleum gas and manual techniques. 1 Dismantling is carried out directly onshore, producing hazardous materials that can pollute the environment. These materials include polycyclic aromatic hydrocarbons, polyvinyl chloride, polychlorinated biphenyl, heavy metals, and tributyltin. 2 Hydrocarbon compounds usually originate from tanks, pipes, machines, ballast water, waste fuel, oil, and grease produced during the cleaning stage. 3 In addition to marine pollution, soil and sediment quality has also decreased due to hydrocarbons and metals.
Bioremediation is a soil remediation technique which uses microorganisms to degrade soil contaminants through biological activities. There are three general principles in bioremediation practice, including stimulation of the indigenous soil bacteria (biostimulation), such as nutrient Research amendment and supplying oxygen (aeration); exogenous bacteria additions (bioaugmentation); and intrinsic or natural attenuation with requirements, such as a high population of microorganisms, nutrient bioavailability, and environmental conditions, which lead to organism growth and efficient contaminant degradation. 4-6 Studies show that biostimulation, bioaugmentation, or the combination of the two are effective in reducing pollutant concentrations in the soil, especially hydrocarbons. 7-9 Previous research succeeded in isolating Bacillus subtilis from diesel-contaminated seawater on Kenjeran Beach, and Acinetobacter lwoffii from diesel-contaminated soil at a ship dismantling site in Tanjungjati, Bangkalan. 10,11 B. subtilis is capable of degrading hydrocarbons and can survive in the presence of metals. 12- 14 In addition, A. lwoffii is a soil microorganism with the ability to decompose hydrocarbons. 15, 16 Currently, there are no studies on the use of these bacteria in bioaugmentation applications to soil contaminated with hydrocarbons and metals. The purpose of the present study was to determine the bioremediation potential to total petroleum hydrocarbons (TPH) and metals from ship dismantlingcontaminated soil. The study uses a combination treatment of surfactant solution, bioaugmentation (a consortium of Bacillus subtilis and Acinetobacter lwoffii), and biostimulation (nutrient amendment and intermittent aeration).

Soil physical-chemical analysis
Soil parameters, such as pH, water content, organic carbon, total nitrogen, total phosphorus, phosphorus available, total potassium, texture,  Research and metals bioavailability (iron (Fe), manganese (Mn), lead (Pb), cadmium (Cd) bioavailable) were analyzed according to the Indonesian technical guidelines for soil chemical analysis. 17 Afterward, the samples were agitated with ethylenediaminetetraacetic acid 0.05 N solvent (pH adjust 7.00) for 1 hour and filtrated prior to atomic absorption spectrophotometry analysis (Agilent 240FS AA, Santa Clara, CA). The soil was n-hexane solvent extracted using the ultrasonic water bath method for TPH analysis. 18 Ten (10) g of soil, anhydrous sodium sulfate, and 35 mL n-hexane were placed in a 100 mL bottle (Duran, Germany). Extractions were conducted for 60 minutes, at 50°C in an ultrasonic water bath (Krisbow, Indonesia). The extract was filtrated with glass wool and 25 mL n-hexane was added and made up to 60 mL final volume of supernatant, which was put into a flask and kept in a fume hood for 3-4 days.

Bacteria preparation consortium
Bacillus subtilis and Acinetobacter lwoffii have been evaluated in the previous studies. 10, 11 The strains were inoculated in selective media Bacillus differentiation agar (Himedia, India) and Herellea agar (Himedia, India), and incubated for 24 hours in a 37°C incubator (Memmert, United Kingdom). Afterward, both the B. subtilis and A. lwoffii were inoculated in nutrient broth (Merck, Germany) and placed on a shaker with 150 rpm at room temperature (28 + 0.2°C) for 13 and 8 hours, respectively. Media selective for A. lwoffii and B. subtilis were used to determine both bacteria species with the pour plate technique during the bioremediation process.

Statistical analysis
The effects of bioremediation on the concentrations of TPH, Fe, and Mn were determined via one-way analysis of variance using the Statistical Package for the Social Sciences software (SPSS) version 21. Parameters of pH, CO 2 concentration, and total colony number were analyzed using Spearman correlation.

Figure 2 -Reactor design for bioremediation experiment
Research Results Table 1 shows that both samples were composed of slightly alkaline, sandy loam soil with low nutrient content, except for phosphorus compounds, compared to data interpretation of the Indonesian technical guidelines for soil chemical analysis. 17 Critical limit or level is defined as the maximum acceptable concentration of metals without long-term effects on the ecosystem. The critical limit of soil is also related to its impact on organisms and plants. 19 Maize grown on soils with Fe and Mn approaching the critical limit had the highest accumulation of metals. However, maize's metal absorption ability was decreased when grown on soil which exceeded the critical limit.  Figure 3 shows the highest CO 2 concentrations in 1AV and 2AV had been achieved in the first 7 days of incubation, while the concentration of A. lwoffii and B. subtilis colonies in both reactors increased until the 28 th day. However, they started to decrease until the end of the bioremediation experiment, which was on day 42. In contrast, the highest CO 2 concentrations in the 1TV and 2TV reactors was achieved on the 21 st day. The largest concentration of bacterial colonies occurred on the 14 th day and 28 th day in 1TV and 2TV reactors, respectively, followed by a decrease on the 42 nd day. After 21 days, the concentration of CO 2 decreased on 1AV, 1TV, 2AV, and 2TV reactors, even though there was no significant difference between the 28 th day and the 42 nd day. In reactors that only received intermittent aeration treatment without bacterial consortium, the CO 2 concentrations and the concentration of A. lwoffii and B. subtilis colonies did not change significantly from the beginning to the end of the bioremediation experiment, and the value was almost the same as in the control reactor (soil 1=1T and soil 2 = 2T).
In 1AV, 1TV, 2AV, and 2TV reactors, a surfactant solution was applied to the sample before the addition of a bacterial consortium. Figure 3 shows The 1AV and 2AV reactors had acidic soil pH in the first 2 weeks, which rose close to 7 on the 21 st day ( Figure  4). Furthermore, 1AV reactor had a neutral and stable pH until the 42 nd day. However, the soil pH of 2AV reactor decreased to 5.8 on the 28 th day, then became neutral on the 35 th to 42 nd day. The soil pH in 1TV and 1A reactors decreased until the 28 th day, and neutral conditions began to be evident from the 35 th day, a condition that also occurred in 2A reactor. The soil in 2TV reactor changed significantly on the 7 th day, then increased to 6.3 on the 14 th day before dropping to pH 5.8 on the 21 st day. During the 4 th week, the soil pH of 2TV reactor increased to neutral on the 35 th day. In the control reactor (1T, 2T), soil pH was never lower than 6.5, and between the 5 th week and the end of the bioremediation experiment, soil pH was 7.
There was a correlation between CO 2 concentration, pH, and A. lwoffii Nadhirawaty, Titah Research and B. subtilis colony number. There was a significant negative correlation between CO 2 concentration and pH (r = -0.644; p < 0.05). This indicated that the increase of CO 2 concentration was always followed by a decrease in pH value during bioremediation ( Figure  4). A negative correlation was also evident between pH for both species of bacteria colony numbers. Total colony number of A. lwoffii to pH value was r = -0.288 (p > 0.05) and B. subtilis to pH value was r = -0.083 (p > 0.05). This indicated that the increase in colony number of both species followed a decrease in pH value. Figure 5 represented  (1TV, 1T, 2TV, 2T).

Discussion
The present study sought to determine whether the combination of surfactant solution, bioaugmentation, and biostimulation has the potential to decrease the concentration of TPH, Fe, and Mn in soil contaminated by ship dismantling activities in Tanjungjati Village, Bangkalan district, Indonesia.
In both Soil 1 and Soil 2 samples, the levels of TPH, Fe, and Mn exceeded recommended maximum levels. The lower nutrient content and higher organic carbon of Soil 2 was due to its higher hydrocarbon compound content. The presence of hydrocarbon compounds in soil could increase organic matter, carbon, nitrogen, and also decrease the level of phosphorus, magnesium, calcium, sodium, and potassium. 24,25 Moreover, the presence of hydrocarbon compounds in soil pores decreased water holding capacity and was characterized by lower water holding capacity in Soil 2 compared to Soil 1.